The present invention relates to the determination of amylase.
.alpha.-Amylase (1,4-.alpha.-D-glucan glucanohydrolase, EC 3.2.1.1) catalyses the random endohydrolysis of 1,4-.alpha.-glucosidic linkages in starch, glycogen and other .alpha.-glucose polymers. It is secreted by the pancreas into the digestive tract where it is involved in the hydrolytic breakdown of food polysaccharides ultimately to produce glucose.
The determination of .alpha.-amylase is important in the diagnosis of acute and chronic pancreatitis. These disorders are commonly caused by alcohol abuse or biliary tract diseases. Typically, plasma amylase levels increase five-fold or more over basal levels.
Known methods for amylase determination include amyloclastic, saccharogenic, and chromogenic methods. The amyloclastic methods measure the breakdown of a starch substrate by viscosimetric, turbidimetric iodometric, or nephelometric procedures. They are not frequently used.
The saccharogenic methods measure the formation of sugars by their reducing ability or by specific enzymic methods. The latter include colorimetric and spectrophotometric coupled-enzyme methods involving the measurement of specific chromophores. Many saccharogenic methods suffer from the drawback of having starch as substrate, which does not give reproducible results.
In the chromogenic methods, synthetic substrates are used which yield coloured products that can be measured spectrophotometrically. Their simplicity, sensitivity, suitability for continuous monitoring and convenient automation account for their current popularity.
Examples of chromogenic substrates include dyes covalently attached to an insoluble polysaccharide backbone. The action of the amylase releases soluble dye fragments which can be measured spectrophotometrically after removing unhydrolysed substrate. A disadvantage of this approach is the difficulty in adapting the determination to automatic analysers.
Other chromogenic substrates include p-nitrophenyl oligosaccharides, for example of the general formula PNP-Gn, where PNP represents a p-nitrophenyl group. G represents a glucose residue, and is from 5 to 7. PNP-G5, PNP-G6 and PNP-G7 have all been used in commercial reagents and have become increasingly popular in clinical practice (see, for example, U.S. Pat. No. 4,233,403). Hydrolysis of these substrates by the action of amylase yields a mixture of smaller p-nitrophenyl oligosaccharides. Further hydrolytic degradation of these products, catalysed by .alpha.-glucosidase releases the coloured p-nitrophenolate anion which may be determined spectrophotometrically at 405 nm.
The use of the p-nitrophenyl oligosaccharides suffers from a number of drawbacks. They are sensitive to pH, temperature and protein concentration, and they have a lag-phase of around 5 minutes before absorbance readings for a kinetic assay may be accumulated. A major problem is that the reagent mixtures are only stable in solution for a short period.
A variation of this type of assay has recently emerged which appears to offer a shorter lag-phase and longer reagent stability in solution, as described in Clin Chem, 33, 524 (1987). The method uses a blocked p-nitrophenyl maltoheptaoside substrate together with two indicator enzymes; glucoamylase to cleave further the amylase reaction products, and a-glucosidase to release p-nitrophenolate. The blocking group (benzylidene) at the non-reducing end of the oligosaccharide renders the substrate resistant to cleavage by glucoamylase (an exoglycosidase) or glucosidase, but not to amylase (an endoglycosidase). This method probably represents one of the better methods available for amylase determin- ation using solution reagents, though it still takes a total of 4 to 5 minutes.